Molecular interaction analysis of ß-1, 3 glucan binding protein with Bacillus licheniformis and evaluation of its immunostimulant property in Oreochromis mossambicus.

Analyzing the health benefits of any two immunostimulants (synbiotics) in combined form and information on their interactions gain more visibility in the usage of synbiotics in aquafarms. With this intention, the current work explores the immunostimulant effect and structural interaction of synbiotic (ß-1, 3 glucan binding protein from marine crab, Portunus pelagicus (Ppß-GBP) and Bacillus licheniformis) on Oreochromis mossambicus. The experimental diet was prepared with Ppß-GBP and B. licheniformis, and nourished to the fingerlings of O. mossambicus for 30 days. After the experimental trial, a higher growth rate and immune reactions (lysozyme, protease, myeloperoxidase and alkaline phosphatase activity) were noticed in the fish nourished with synbiotic (B. licheniformis and Ppß-GBP) enriched diet. Moreover, the synbiotic enriched diet elevated the antioxidant responses like glutathione peroxidase (GSH-Px) and catalase (CAT) activity in the experimental diet-nurtured fish. At the end of the feed trial, synbiotic diet nourished fish shows an increased survival rate during Aeromonas hydrophila infection, reflecting the disease resistance potential of experimental fish. Also, the interaction between Ppß-GBP and Bacillus licheniformis was analyzed through computational approaches. The results evidenced that, Ppß-GBP interacts with the B. licheniformis through sugar-based ligand, ß-glucan through a hydrogen bond with a good docking score. Thus, the synbiotic diet would be an effective immunostimulant to strengthen the fish immune system for better productivity.

ß-1,3-Glucan binding protein-based silver nanoparticles enhance the wound healing potential and disease resistance in Oreochromis mossambicus against Aeromonas hydrophilla.

Here we attempted to synthesize ß-1,3-glucan binding protein-based silver nanoparticles (Phß-GBP-AgNPs) and evaluate its wound healing and disease resistance prompting ability in Oreochromis mossambicus. Using a column chromatography technique, an immune molecule, Phß-GBP was purified from the haemolymph of rice field crab, Paratelphusa hydrodromus. Phß-GBP-AgNPs were synthesized and described through SDS-PAGE, UV-vis spectroscopy, HR-TEM, XRD and FTIR analysis. HR-TEM revealed that the synthesized Phß-GBP-AgNPs were spherical with a 20-40 nm size range and the particles were not aggregated. Wound and infection were experimentally generated in O. mossambicus and treated with Phß-GBP, chem-AgNPs and Phß-GBP-AgNPs for 20 days. The immune parameters (peroxidase, lysozyme and protease) and antioxidant enzymes (SOD and catalase) were examined in the serum of experimental fish. Phß-GBP-AgNPs elevated the immune and antioxidant enzymes during the healing process and enhanced the wound healing percentage in fish than Phß-GBP and chem-AgNPs. The immune parameters and antioxidant enzymes were declined in the serum of fish (treated with Phß-GBP-AgNPs) after the mid-period of wound healing. Compared to others, relative percentage survival was increased in experimentally wounded and infected fish treated with Phß-GBP-AgNPs against Aeromonas hydrophila. Moreover, Phß-GBP-AgNPs exhibited less toxicity towards Artemia salina than chem-AgNPs during 24 h exposure period. As a result, Phß-GBP-AgNPs may act as an alternative to commercial antibiotics and be considered an effective immunostimulant in treating skin lesions in intensive farming.

Protective activity of beta-1, 3-glucan binding protein against AAPH induced oxidative stress in Saccharomyces cerevisiae.

The present work aimed to evaluate the antioxidant efficacy of beta-1,3-glucan binding/recognition protein against oxidative stress-induced Saccharomyces cerevisiae. Beta-1,3-glucan binding/recognition protein was attained from the Paratelphusa hydrodromus (Phß-GBP) using laminarin coupled Sepharose CL-6B column. The structural characteristics of Phß-GBP were analyzed through circular dichroism (CD), fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance spectroscopy (1H NMR) analysis. CD spectrum showed the occurrence of α-helix (4.5%), ß-sheets (23.6%), ß-turn (17.2%) and random coils (54.8%). FTIR confirms the occurrence of amide and aromatic compounds whereas 1H NMR predicts the secondary structures and presence of amino acids in the Phß-GBP. In vitro radical scavenging analysis disclose that Phß-GBP has the potential to scavenge DPPH (73%), peroxyl radicals (81%) and hydrogen peroxide (56%) at 100µg/ml concentration. Reactive oxygen species production, lipid peroxidation, cell death, and DNA damage were decreased in the Phß-GBP pretreated S. cerevisiae. In silico protein-protein interaction was performed between the ß-GBP-glutathione reductase and ß-GBP-catalase A. The interaction study reveals that glutathione reductase and catalase A interacts with ß-GBP to reduce the oxidized glutathione and remove free radicals. This finding demonstrates that Phß-GBP may act as a good antioxidant which protects from human pathologies linked with oxidative stress.

ß-1, 3 glucan binding protein based selenium nanowire enhances the immune status of Cyprinus carpio and protection against Aeromonas hydrophila infection.

In the present study, immunoenhancing effect of ß-1, 3 glucan binding protein based selenium nanowire (Phß-GBP-SeNWs) in common carp, Cyprinus carpio was assessed. Biological based selenium nanoform was synthesized, using crustacean immune molecule ß-GBP purified from the haemolymph of Paratelphusa hydrodromus. The morphological property of Phß-GBP-SeNWs was analyzed through TEM which reveals, the synthesized nanowire exhibits approximately 30-50 nm width with smooth surface. For this current study, fish were fed with experimental diet includes Phß-GBP, sodium selenite, selenomethionine and Phß-GBP-SeNWs supplemented diet at different concentrations (0.5 mg, 1 mg and 2 mg) for 30 days. The growth performance, cellular and humoral immune responses (myeloperoxidase, reactive oxygen species, alkaline phosphatase and lysozyme activity) and antioxidant enzymes (glutathione peroxidase and catalase activity) in the fish fed with Phß-GBP-SeNWs supplemented diet were significantly increased in dose-dependent manner, which was observed at two different interval period (15th and 30th day). Also, Phß-GBP-SeNWs supplemented diet fed fish gain resistant after challenged with aquatic pathogen Aeromonas hydrophila and the relative survival percentage was increased. Agar disc diffusion and BacLight assay clearly demonstrated the antibacterial property of plasma of fish fed with Phß-GBP-SeNWs supplemented diet against aquatic pathogen A. hydrophila, Vibrio parahaemolyticus and Vibrio alginolyticus. Moreover, confocal laser scanning microscopic analysis clearly showed that, Phß-GBP-SeNWs supplemented diet fed fish plasma was more efficient in disrupting the architecture of bacterial colonies and thereby reduced the thickness of biofilm. Thus, the present study indicates that, incorporation of Phß-GBP-SeNWs in the diet enhances the fish immune responses and disease resistance against aquatic pathogens.

Searching for crab-borne antimicrobial peptides: Crustin from Portunus pelagicus triggers biofilm inhibition and immune responses of Artemia salina against GFP tagged Vibrio parahaemolyticus Dahv2.

Marine organisms represent a huge source of novel compounds for the development of effective antimicrobial drugs. The present study focus on the purification of the antimicrobial peptide crustin from the haemolymph of the blue swimmer crab, Portunus pelagicus, by blue Sepharose CL-6B matrix assisted affinity column chromatography. Crustin showed a single band with a molecular mass of 17 kDa in SDS-PAGE analysis. The XRD analysis exhibited peaks at 32° and 45° while a distinct peak with a retention time of 1.8 min resulted in high performance liquid chromatography (HPLC) pointing out the crystalline nature and purity of crustin, respectively. Crustin purified from P. pelagicus (Pp-Cru) showed immunological activities, triggering encapsulation, phagocytosis on Sepharose beads and yeast (Saccharomyces cerevisiae) respectively. Furthermore, encapsulation of GFP tagged V. parahaemolyticus in Artemia salina and challenging study were assessed under CLSM and the potential of Pp-Cru was examined in vivo. In addition, the growth reduction and biofilm inhibition potential of Pp-Cru on Staphylococcus aureus, Enterococcus faecalis (Gram- positive bacteria) and Pseudomonas aeruginosa, Escherichia coli (Gram-negative bacteria) was evidenced by inverted and confocal laser scanning microscopic analysis, revealing that 100 µg/ml of Pp-Cru can disrupt the biofilm matrix thereby the thickness of biofilm was significantly reduced. Overall, the present investigation might provide a sensitive platform to realize the significant function of Pp-Cru in crustacean immune mechanism as well as its potential to bacterial growth inhibitor. The functional properties of purified Pp-Cru antimicrobial peptide may lead to a superior understanding of innate immune response in P. pelagicus species, which suggest the promising application for drug development in aquaculture.

Effect of ß-1, 3 glucan binding protein based zinc oxide nanoparticles supplemented diet on immune response and disease resistance in Oreochromis mossambicus against Aeromonas hydrophila.

Recently, several immunostimulants such as ß-glucan, microbial and plant products have been used as dietary supplements to combat disease outbreaks in aquaculture. The present study investigates the potential of Portunus pelagicus ß-1, 3 glucan binding protein based zinc oxide nanoparticles (Ppß-GBP-ZnO NPs) supplemented diet on growth, immune response and disease resistance in Mozambique tilapia, Oreochromis mossambicus. The immune-related protein ß-GBP was purified from the haemolymph of P. pelagicus using Sephadex G-100 affinity column chromatography. Ppß-GBP-ZnO NPs was physico- chemically characterized and experimental feed was formulated. Fish were separately fed with commercial diet (control-group I) and Ppß-GBP (group II, III, IV), Ppß-GBP-ZnO NPs (group V, VI, VII), chem-ZnO NPs (VIII, IX, X) mixed diet at the concentration of 0.001%, 0.002% and 0.004% respectively. Triplicate groups of O. mossambicus were fed with experimental diets twice a day for 30 days. Fish receiving Ppß-GBP-ZnO NPs supplemented diet showed a significant increase (P < 0.05) in growth performance. Cellular immune responses (myeloperoxidase activity, lysozyme activity and reactive oxygen species activity) and humoral immune responses (complement activity, antiprotease activity and alkaline phosphatase activity) were evaluated at an interval of 15 days during the feeding trial. Results demonstrate that both cellular and humoral immune responses were substantially increased (P < 0.05) in fish fed with 0.004% of Ppß-GBP-ZnO NPs supplemented diet than others. Antibiofilm potential of Ppß-GBP-ZnO NPs against Aeromonas hydrophila was visualized through confocal laser scanning microscopy (CLSM), which reveals reduction in the preformed biofilm thickness to 10 µm  at the concentration of 50 µg/ml. Furthermore, after 30 days of feeding trial, fish were challenged with aquatic fish pathogen A. hydrophila (1 × 107 cells ml-1) through intraperitoneal injection. Challenge study displayed a reduced mortality rate in fish fed with diet containing Ppß-GBP-ZnO NPs. Thus our study suggests that dietary supplementation of Ppß-GBP-ZnO NPs at 0.004% may have a potential effect to enhance the immune system and survival of O. mossambicus.

Phenoloxidase activation, antimicrobial, and antibiofilm properties of ß-glucan binding protein from Scylla serrata crab hemolymph.

In this study, we purified ß-GBP from hemolymph of Scylla serrata crabs using affinity chromatography. The purified S. serrata ß-GBP (Ss-ß-GBP) had 100kDa molecular mass in the SDS-PAGE. MALDI-TOF/TOF analysis was conducted, revealing that the purified 100kDa protein had 96% similarity with ß-GBP of Astacus leptodactylus. Ss-ß-GBP was characterized using high-performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which confirmed the structure of the Ss-ß-GBP. The purified Ss-ß-GBP was functionally analyzed by yeast agglutination and phagocytic reaction assays. Moreover, the PO enhancing ability of Ss-ß-GBP was evidenced through PO activity. Specifically, the antibacterial activity of the Ss-ß-GBP against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was evaluated by determining its minimum inhibitory concentration (MIC)<60µg/ml for all tested species. Furthermore, the antibiofilm efficacy of Ss-ß-GBP at 50 and 100µg/ml was outlined using light microscopy and confocal laser scanning microscopy (CLSM). Bacterial viability assays also outlined the dose-dependent activity of Ss-ß-GBP based on the ratio of live/dead bacterial cells. The results of this study revealed that crab-borne Ss-ß-GBP might be widely used to suppress the growth of pathogenic bacteria.

Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus.

The present study evaluated the dietary supplementation of probiotic Bacillus licheniformis Dahb1 on the growth performance, immune parameters and antioxidant enzymes activities in serum and mucus as well as resistance against Aeromonas hydrophila in Mozambique tilapia Oreochromis mossambicus. Fish (24 ±â€¯2.5 g) were fed separately with three diets, 1) commercial diet (control), 2) diet containing probiotic at 105 cfu g-1 (D1) and 3) diet containing probiotic at 107 cfu g-1 (D2) for 4 weeks. Growth performance in term of final weight (FW) specific growth rate (SGR) and feed conversion ratio (FCR), immune parameters of total protein (TP), alkaline phosphatase (ALP), myeloperoxidase (MPO), lysozyme (LYZ), reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidant parameters of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in serum and mucus were evaluated after 2nd and 4th weeks. The FW, SGR, and FCR of fish fed with D1 and D2 significantly improved (p < 0.05). The activities of ALP, LYZ and MPO in the mucus were significantly higher (p < 0.05) in fish that fed D1 and D2. The TP, ROS, RNS, SOD and GPx in the serum were significantly higher (p < 0.05) in fish that fed D1 and D2. In addition, the challenge test showed that fish fed D1 and D2 enhanced significantly (p < 0.05) the resistance against A. hydrophila (1 × 107 cells ml-1). In conclusion, probiotic B. licheniformis Dahb1 can be applied in diet at 107 cfu g-1 to improve healthy status and resistance against A. hydrophila in tilapia farming.

Biological synthesis of silver nanoparticles using ß-1, 3 glucan binding protein and their antibacterial, antibiofilm and cytotoxic potential.

The present study reports the biological synthesis of silver nanoparticles using crustacean immune molecule ß-1, 3 glucan binding protein (ß-GBP) purified from the haemolymph of blue swimmer crab Portunus pelagicus. The characterization of synthesized ß-GBP based silver nanoparticles (Ppß-GBP-AgNPs) was made by UV-Vis spectroscopy, XRD, FTIR and TEM analysis. UV-Vis spectra recorded the strong absorbance peak at 420 nm due to its surface plasmon resonance. The XRD analysis revealed the crystalline nature of synthesized nanoparticles with Bragg's reflection peaks at (111), (200), (220), (311) planes. FTIR analysis showed the possible functional groups at 3422, 2926, 2847, 1648, 1556, 1407, 1016 and 669 cm-1. The mean particle size of Ppß-GBP-AgNPs was 33-47 nm revealed by TEM analysis. Ppß-GBP-AgNPs exhibit appreciable antibacterial activity against Enterococcus faecalis and Pseudomonas aeruginosa when compared to chemical based AgNPs (Chem-AgNPs). The antibiofilm property of Ppß-GBP-AgNPs was assessed through light microscopy and confocal laser scanning microscopy analysis (CLSM), which clearly demonstrates, thickness of E. faecalis and P. aeruginosa preformed biofilm was reduced to 11 µm & 8 µm from 47 µm & 45 µm respectively. Moreover, exopolysaccharide (EPS) quantification and cell surface hydrophobicity (CSH) index exhibited that, Ppß-GBP-AgNPs had the potential to disturb structural integrity of biofilm by upset EPS matrix and bacterial adhesion to hydrocarbons. In addition, the cytotoxic effect of Ppß-GBP-AgNPs was evaluated against human cervical cancer cells (HeLa). Ppß-GBP-AgNPs effectively inhibit the viability of HeLa cells at 50 µg/ml concentration and the morphological changes in Ppß-GBP-AgNPs treated HeLa cells were observed under phase contrast microscopy.

A study on ß-glucan binding protein (ß-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus.

The present study reports the purification of novel immune molecule ß-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiß-GBP). The purified Fiß-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiß-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for ß-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiß-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiß-GBP had the specific binding site for soluble or insoluble ß-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiß-GBP activating PO were 40°C and pH 7-8. Moreover, even at 100°C Fiß-GBP enhanced PO activity highlighting that Fiß-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiß-GBP significantly promoted the PO activity in presence of Mg2+ and Ca2+. The breakdown of para nitroanilide from Nα-Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiß-GBP and also increased concentration of Fiß-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.

Multipurpose efficacy of ZnO nanoparticles coated by the crustacean immune molecule ß-1, 3-glucan binding protein: Toxicity on HepG2 liver cancer cells and bacterial pathogens.

The effective treatment of cancer and bacterial pathogens are two key challenges in modern nanomedicine. Here, zinc oxide nanoparticles (ZnO NPs) were fabricated using the crustacean immune molecule ß-1, 3- glucan binding protein (Phß-GBP, 100kDa) purified from the heamolymph of Paratelphusa hydrodromus. ß-GBP coated zinc oxide nanoparticles (Phß-GBP-ZnO NPs) were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and high resolution-transmission electron microscopy (HR-TEM) analyses. Phß-GBP-ZnO NPs inhibited the growth of Staphylococcus aureus and Proteus vulgaris. Protein and nucleic acid leakage assays showed that Phß-GBP-ZnO NPs facilitate membrane permeability leading to cell death. The antibacterial activity of Phß-GBP-ZnO NPs was due to the high level of reactive oxygen species (ROS) release from bacterial cells post-treatment with 75µg/mL of Phß-GBP-ZnO NPs. Confocal laser scanning microscopy pointed out that biofilm thickness was highly reduced post-treatment with nanoparticles. Cytotoxicity on human liver carcinoma (HepG2) cells highlighted that 75µg/mL of Phß-GBP-ZnO NPs inhibited viability of HepG2 cells. Phase contrast microscopy showed key morphological changes of HepG2 cells post-treatment with Phß-GBP-ZnO NPs. Overall, Phß-GBP-ZnO NPs can be further considered for the development of novel drugs against microbial pathogens and HepG2 cells.

Role of purified ß-1, 3 glucan binding protein (ß-GBP) from Paratelphusa hydrodromus and their anti-inflammatory, antioxidant and antibiofilm properties.

ß- 1, 3-glucan binding protein (ß-GBP), a pattern recognition protein (PRP), plays a critical role in triggering the innate immune response by detecting ß-glucan found on the surface of microbes. In the present study, ß-GBP was purified from the haemolymph of rice field crab Paratelphusa hydrodromus by affinity column chromatography. The monomeric protein Ph-ß-GBP appeared as a single band with a molecular weight of approximately 95 kDa in SDS-PAGE analysis and its purity was determined to be 89% by HPLC. MALDI-TOF/TOF analysis revealed that, the purified 95 kDa protein display 36% similarity with ß-GBP of crayfish Astacus lepidodactylus. Purified Ph-ß-GBP exhibited increased agglutination, phagocytic activity and encapsulation in a dose-dependent manner, indicating the involvement of Ph-ß-GBP in cellular immune response against pathogens in crustaceans. Moreover, addition of Ph-ß-GBP increased the prophenoloxidase (proPO) and serine protease activity, possibly contributing to the clearance of pathogens. The antioxidant activity of Ph-ß-GBP was determined by DPPH radical scavenging activity demonstrates maximum scavenging activity of 78.4%. In addition, RBC membrane stabilization and inhibition of protein (albumin) denaturation proved anti-inflammatory property of Ph-ß-GBP. Furthermore, light microscopic and confocal laser scanning microscopic analysis revealed that the reactive compound (laminarin and Ph-ß-GBP) reduced the biofilm thickness of Gram-positive (Enterococcus faecalis) and Gram-negative (Vibrio parahaemolyticus) bacteria at the concentration of 25 µg/ml. Taken together, our results demonstrate that, the ß-GBP triggers proPO activating system in rice field crab P. hydrodromus and plays a vital role in innate defense mechanism against invading pathogens.

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties.

The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp-Lec). The Pp-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 155 kDa in SDS-PAGE. The surface morphology of purified Pp-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X-ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp-Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp-Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp-Lec showed the broad spectrum of antibacterial activity against Gram-positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp-Lec against selective Gram-negative bacteria showed the disruption of biofilm architecture at the concentration of 50 µg ml-1.

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity.

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system.

Multifunctional role of ß-1, 3 glucan binding protein purified from the haemocytes of blue swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product.

ß-1, 3 glucan binding protein (ß-GBP) was isolated from the haemocytes of blue swimmer crab, Portunus pelagicus and purified by laminarin coupled Sephadex G-100 affinity column chromatography. The purified ß-GBP has the molecular mass of 100 kDa, confirmed by SDS-PAGE. The X-ray diffraction analysis of purified ß-GBP indicates the crystalline nature of the protein and also the presence of single peak confirming the existence of ß-glucan molecule. The results of agglutination assay showed that the purified ß-GBP had the ability to agglutinate with yeast cell, Saccharomyces cerevisiae and mammalian erythrocytes. ß-GBP can agglutinate with yeast cells at the concentration of 50 µg/ml. The phagocytic and encapsulation activity of purified ß-GBP from P. pelagicus was determined with yeast cell S. cerevisiae and sepharose bead suspension respectively. This reveals that, ß-GBP have the ability to detect the pathogen associated molecular patterns (PAMP) found on the surface of fungi and bacteria. The recognition of invading foreign substances and in the involvement of functional activities induces the activation of prophenoloxidase. This revealed that ß-GBP play a major role in the innate immune system of crustaceans by stimulating the prophenoloxidase system. Moreover, it was obvious to note that ß-GBP reaction product exhibited antibacterial and antibiofilm activity against Gram positive and Gram negative bacteria. This study concludes the functional aspects of ß-GBP purified from P. pelagicus and its vital role in the stimulation of prophenoloxidase cascade during the pathogenic infection.